The goal of this proposal is to continue our successful development of TOPAS-nBio, a Monte Carlo simulation toolkit specifically designed to connect research disciplines. TOPAS-nBio simulates the initial energy deposition events (physics), then follows the diffusion and reaction of chemical species (chemistry) to infer biological observables at the cell and organelle scale (biology). The developed application already lays the foundation to investigate biological ef- fects of radiation in cell organelles using a mechanistic systems biology modeling approach. However, with constant advances in our understanding of cellular repair processes, the ques- tions asked by the radiation biology community are increasing in complexity. These advances, including more detailed information of various cells lines/types, deficiencies in DNA repair pathways and potential contributions of non-nuclear cell components, need to be considered to correctly describe cell response to radiation damages. Accordingly, in this renewal application, we focus on improving the accuracy of the simulations by including more representative chem- ical reactions and transitioning towards a predictive model that can be applied to specific cell types. To further extend the reach of TOPAS-nBio, we will include changes in the microenvi- ronment across tumor volumes and move towards mechanistic modeling of radiation effects in vivo. Thus, the new developments of TOPAS-nBio will offer predictions of biological outcome from the initial radiation track structure for various cell types for in vitro and in vivo experiments, and thereby drive hypothesis generation at the forefront of bio-physical research. TOPAS-nBio provides an ideal framework to include and test new effect models, cell lines or microenviron- mental conditions. Overall, TOPAS-nBio will continue the mission to advance our under- standing of the fundamental response of tissue to radiation.